Nuclear receptors (NRs) comprise a family of 48 transcription factors, which are regulatory proteins that turn genes on or off. In contrast to othe transcription factors, the activity of NRs is physiologically regulated by small molecules (ligands), including sex hormones, glucocorticoids, vitamins, lipids, and others, which makes these proteins amenable to pharmacological intervention for the treatment of many diseases, including inflammation, cancer, and diabetes. NRs therefore represent one of the most successful classes of therapeutic drug discovery targets. Orphan NRs are receptors for which no physiological ligands were known when they were first identified. This set of NRs remains of enormous medical interest as their physiological roles and possible regulation by small molecules and drugs are still emerging. The objective of this study is to determine high resolution crystal structures of the remaining orphan NR ligand-binding domains (LBDs), to correlate the structures with biological functions of these receptors, and to explore the structura information for drug discovery that targets these receptors. All nuclear receptors contain at least one of two conserved domains: the centrally-located DNA-binding domain (DBD) and the C-terminal LBD. The LBD is the key functional domain that mediates the ligand binding, receptor dimerization, ligand-regulated transcriptional function of nuclear receptors. As such the LBD has been the focus of intense structural studies and pharmaceutical discovery. Crystal structures of most of the human nuclear hormone receptor LBDs have been determined and these structures have provided key mechanisms of ligand regulation and ligand discovery for nuclear receptors. Work from the first renewal of this application identified novel regulatory mechanisms for two repressive orphan NR, SHP and TLX, and suggested that similar mechanisms may be shared by other repressive orphan NRs. In the specific aims of this application, we will test this hypothesis by functionally analyzing the newly discovered regulatory interfaces and by determining the crystal structure of SHP in different states to identify the structural mechanisms by which SHP is regulated by small molecules and by which SHP represses other NRs. Following the structural determination, we will validate the functional significance of key structural elements through close collaborations with Drs Steve Kliewer, David Mangelsdorf, Chun-Li Zhang, and Pat Griffin, who are key experts on these receptors. Significance: The structural information generated in this application will significantly enhance our understanding of the molecular mechanisms of how these orphan nuclear receptors have evolved for their respective ligand-dependent or -independent signaling pathways, and can serve as rational templates for drug discovery that targets these receptors.